Page 45 - Haematologica-April 2018

Hepcidin and hyperferritinemia in Gaucher disease
Table 1. Iron-related biological parameters of the untreated and treated Type 1 Gaucher disease (GD1) cohorts.
Group ERT
Men No
Women No
Children No
Men Yes
Women Yes
Children Yes
Normal values Men
Women Children
N Age (years)
9 20-65
14 19-63
11 3-13
30 17-80
29 17-64
Ferritin (mg/L)
563±148
[53-1443]
518±162
[27-2141]
287 ±62
[97-779]
372±52
[56-1341]
278±88
[33-2572]
104*±19
[52-175]
30-300
15-150 15-150
Fer Transferrin (mmol/L) saturation
(%)
16.0±2.2 23.5±3.3 [8-22] [10.0-35.6]
13.9±1.4 21.7±2.3
[6-20] [10.0-33.6] 10.5±0.6 14.9±1.5
[9-13] [10.8-21.0]
17.5±0.9 28.7±1.7
[8-28] [11.0-48.2] 16.9±1.2 29.8±2.8 [5-34] [8.0-73.0] 16.6±1.0 19.9±2.7
[1-34] [11.0-28.0]
12-26 20-40
10-26 15-35 10-26 15-35
Hepcidin (ng/mL)
12.9±3.6 [2.3-35.1] 6.4±2.1 [0-20.5 6.6±1.2 [1.0-15.0] 24.1±5.1 [1.1-127.7] 10.2±2.5
Hemoglobin (g/dL)
14.1±0.3 [12.9-16.2] 11.9±0.4 [6.4-13.7] 11.3±0.3 [9.5-12.5] 15.5***±0.2 [13.6-17.3] 13.6***±0.2
MCV (fL)
84.8±1.8 [75.0-91.9] 88.2±1.8 [76.0-100.0] 74.1±1.5 [67.0-80.0] 85.1±0.9 [75.0-96.0] 89.3±0.9 [81.0-98.0] 79.7*±1.7 [70.0-83.0]
80-100
7
9-14
[0-46.1] [12.2-16.5]
6.4±2.4 13.2**±0.4
[0-17] [12.1-14.4] 13-18
1-20
12-17 11,5-16
Data are expressed as the means ± Standard Error of Mean,and the range is shown in brackets.The two-tailed Mann-Whitney test was used to compare each subgroup between the untreated and treated patients (*P≤0,05; **P≤0,01; ***P≤0,001). N: number; ERT: enzyme replacement therapy. MCV: mean corpuscular volume; Fer: serum iron.
Cell culture
The J774 cell line derived from mouse macrophages was pur- chased from the American Type Culture Collection (ATCC) and was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with a 10% low-endotoxin fetal bovine serum (FBS), 4 mM glutamine and 1.5 g/L sodium bicarbonate at 37°C with 5% CO2 in the atmosphere. Cells were plated and main- tained overnight in the above medium in a 6-well plate. Confluent cells were first treated with FeNTA (100 mM Fe/400 mM NTA) to drive the expression of FPN at the cell membrane. Twenty-four hours (h) later, 1 mM conduritol B epoxide (CBE) was added for 96 h.
Immunofluorescence and confocal microscopy
Treated cells were washed 3 times with PBS, fixed with 3% paraformaldehyde for 10 min at room temperature, washed again 3 times with PBS, incubated with 20 mmol/L glycine for 10 min, and subsequently permeabilized for 30 min with PBS containing 0.1% saponin. FPN staining was performed using an anti-FPN antibody (Alpha Diagnostic International, San Antonio, USA) (1/50, for 1 h at room temperature). Actin staining was performed using Alexa Fluor 635 phalloidin (1/200, for 30 min at room tem- perature). The coverslips were mounted using Dako glycerol medium supplemented with 2.5% 1,4 diazabicyclo-(2.2.2)octane (DABCO) (Sigma Aldrich, St Louis, MO, USA) as the anti-fading reagent. Confocal images were taken with a high-resolution con- focal microscopy LSM 780 (ZEISS, Marly-Le-Roi, France) equipped with a 63X oil immersion objective.
Statistical analysis
Statistical analysis was performed using Prism 7 software (GraphPad Software, La Jolla, CA, USA). The unpaired Mann- Whitney test and the paired Wilcoxon test were used to compare biological values. The χ2 test was used to compare patient groups. Spearman’s test was performed to qualify the correlations. P<0.05 was considered statistically significant. Other methods are described in the Online Supplementary Appendix.
Results
Biological features of the untreated patients
The main biological parameters are shown in Table 1. As expected, high ferritinemia was found in untreated patients, with mean values of 563, 518 and 287 g/L in the subgroups of men, women and children, respectively. Twenty-two (65%) out of these 34 patients showed high serum ferritin levels (67% of men, 57% of women and 73% of children) (Online Supplementary Table S2). Despite a wide range of values, we found that the serum ferritin level in women was particularly high, with an average value exceeding three times the accepted normal level. Men and women (adult patients) exhibited a low normal level of TS (means of 22%). However, young patients were iron deficient, with a mean TS of 14.9% and decreased serum iron (Table 1). We also measured serum hepcidin levels by liquid chromatographic-tandem mass spectrometry (LC-MSMS). In all groups, the level of serum hepcidin was within the normal range (mean, 12.9±4 ng/mL for men, 6.4±2 ng/mL for women and 6.6±1 ng/mL for children; the normal range is 1-20 ng/mL) (Table 1).
As expected, anemia was observed in 33% of our patients (40% of children, 43% of women and one man, 11%), although the hemoglobin level was only slightly reduced (Online Supplementary Table S3). Anemia was nor- mocytic with respect to a mean corpuscular volume (MCV) of 74.1±1.5 fL in children (Table 1). Soluble trans- ferrin receptor (sTfR) was also slightly increased with a mean of 2.40±0.24 mg/L (normal values ≤1.76 mg/L).
Hyperferritinemia is independent of the global iron status and systemic inflammation
Because ferritin is stimulated during inflammation, we investigated the inflammatory status of our cohort of GD patients. All individuals included in the study were nega- tive for CRP (<5 mg/L). We also measured the serum level
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